1. Field of the Invention
The present invention relates to an improvement in calibration of measured values obtained by analyzing liquid samples. More particularly, it provides a calibration method wherein the measured values distributed in a wide range can be calibrated or corrected even when standard samples or calibrators containing an analyte in the concentration of limited range can be prepared.
2. Description of the Related Art
In the conventional quantitative analysis of an analyte contained in a liquid sample, the content of analyte in a test sample is determined by measuring an optical density (absorbancy or optical density of the transmitting or reflected light) of the liquid sample after it is subjected to a proper chemical or enzymatic reaction. In such a method, it is a common practice to determine the content of analyte by using a standard curve (commonly referred to as "calibration curve") which has been preliminarily drawn by plotting the interrelation between the known contents of analyte in the standard samples and the optical densities of the standard samples.
However, a number of standard samples must be prepared and examined to draw a standard curve, and thus cumbersome and time-consuming operations are needed therefor. Particularly when a liquid sample obtained from a living body, such as whole blood, plasma or serum, is used as in the case of a clinical assay, it is difficult to prepare always many standard samples (body fluids) each containing a known content of an analyte. To obviate such a difficulty, it has been proposed and practised to prepare a tentative standard curve which is corrected or calibrated by finding the difference or error from the standard curve. In detail, only a few standard samples or calibrators are examined to find measured values which are used to correct the standard curve to prepare a calibration curve which is used for the determination of real measured values of respective samples.
For example, calibrators containing standard values (calibration values; The term "calibration value" used throughout the specification means the given value for which calibration is to De made, and may be referred to as "real value" or "correct value") of H, M and L are subjected to the same assaying procedure to find optical densities or other proper parameters from which real measured values h, m and l are found by referring to the standard curve. The interrelation between the standard values H, M and L and the real measured values h, m and l are plotted to draw a calibration curve as shown in FIG. 2, and the measured value of each examined sample is corrected by using the thus drawn calibration curve. In an automated analyzer, the function of the calibration curve (generally in the form of a quadratic equation) is determined from the data (l, L), (m, M) and (h, H) by the least squares method to obtain the following correction equation of: EQU Y=.alpha.+.beta.X+.gamma.X.sup.2
Then, the measured value is corrected by using the thus obtained correction equation. When the differences in content between the standard values (calibration values) L, M and H are sufficiently large as is the case illustrated in FIG. 2, the effect of curvature of the calibration curve (i.e. the contribution of the term of second order) is small even if the measured values l, m and h are varied independently, so that the measured values distributed in a wide range can be appropriately corrected by the use of the calibration curve.
However, if the differences in content of the standard correct values L, M and H are not large enough, the correct value above the uppermost standard value H and the correct value below the lowermost standard value L tend to contain large errors. Particularly when the measured values l, m and h are varied in the manner as shown in FIG. 3, the calibration curve has an extreme value within the determination range to make it impossible to correct the measured values.
Such a problem arises, for example, in a dry analysis method wherein a dry analysis element is used to analyze an analyte in a liquid sample taken from a living body fluid, such as whole blood, plasma or serum.
The dry analysis element is generally composed of plural layers including, for example, a reagent layer, a porous spreading layer, etc., laminated sequentially on a transparent support. An aqueous liquid sample is spotted on the spreading layer to migrate into the reagent layer which is colored by the action of the analyte contained in the sample. The density of the colored reagent layer is determined by measuring the optical density of the reflected light, and the quantity or content of the analyte contained in the sample is determined by means of colorimetric method. Specific examples of dry analysis element are disclosed in U.S. Pat. Nos. 2,846,808, 3,036,893, 3,368,872 and 3,992,928, Unexamined Japanese Patent Publication Nos. 53888/1974 (corresponding to U.S. Pat. No. 3,992,158), 164356/1980 (corresponding to U.S. Pat. No. 4,292,272), 222769/1985 (corresponding to EP 0 162 302A), 4959/1986 (corresponding to EP 0 166 365A) and 90859/1980 (corresponding to U.S. Pat. No. 4,258,001), Clinical chemistry, 24, 1335-1350, (1978), Analytical Chemistry, 55 (4), 498-514, (1983 ) and Clinical Chemistry, 27, 1287-1290, (1981).
The liquid sample spotted on the spreading layer spreads on the spreading layer to cover a generally circular zone having an area substantially in proportion to the volume of the spotted sample, and then liquid ingredients migrate into the reagent layer while the solid ingredients are filtered off. As a result, a substantially constant volume of aqueous liquid sample is fed to each unit area of the reagent layer. This function is known as a spreading function or metering function. However, the spreading function is significantly affected by the properties viscosity, specific gravity, pH, etc.) of the spotted aquaeous liquid sample. Accordingly, when an analyte contained in a body fluid, such as whole blood, plasma, serum or urine, is to be analyzed, it is preferable to use a calibrator which has properties resembling the body fluid taken from a living body. For example, when albumin or a total protein in blood is quantitatively analyzed, used calibrators are prepared by dissolving lyophilized human blood serum to have proper standard contents. However, the properties of the calibrators are significantly differentiated from those of the body fluids of natural origin as the content of albumin or total protein is varied
in a wide range. For this reason, the difference between the lowermost content of analyte and the uppermost content of analyte in the calibrators cannot be set large enough to cover a wide determination range. As a result, when a calibration curve is drawn by using the least squares method, adequate calibration cannot be made within a wide range since an extreme value is found in the thus drawn calibration curve at the worst case.